The five classical groups of insecticides are: 1) inorganic stomach poisons based on heavy metals, such as mercury, arsenic, zinc and lead; 2) respiratory blocking agents, such as oils and soaps; 3) natural contact insecticides, such as pyrethrum, rotenone and nicotine; 4) volatile compounds, such as p-dichlorobenzene, hydrogen cyanide and hydrogen sulfide and 5) a wide range of modern synthetic insecticides that act on different systems in the insect. These synthetic insecticides include the various halogenated organic materials, such as DDT, Aldrin, methyl bromide, etc., and the nerve toxins such as malathion and parathion. Each of these groups has advantages and disadvantages which are well known to those skilled in the art. The disadvantage of most concern in using many of these, except perhaps for the soaps and some of the oils and natural insecticides, is their toxic effects on mammals.
During the last thirty years a new class of stomach poisons has appeared which has the advantage of being toxic to certain insects but harmless to mammals. This new class of toxins is based on proteins which the cells of Bacillus thuringiensis synthesize during their spore forming growth phase. When the larvae of certain insects, especially the larvae of the cabbage butterfly, eat leaves contaminated by these proteins, these proteins act as potent endotoxins.
Recently Ellar et al., (U.S. Pat. No. 4,918,006), incorporated herewith by reference, using genetic engineering techniques, have been able to induce the cells of E. coli and B. subtilis cultures to produce large amounts of one of the several proteins synthesized by B. thuringiensis. This protein can be readily isolated, dissolved in suitable organic solvents, combined with wetting agents and sprayed upon plants. These preparations act as stomach poisons. They are also toxic to mosquito larvae.
Even more recently research workers at the University of Wisconsin have been able by genetic engineering techniques to introduce the plasmids containing the DNA code for this B. Thuringiensis protein into russet potato plants. Such plants produce the toxic protein in their leaves. Thus, no need exists for the grower to spray his plants for the control of potato beetles since the leaves are now toxic to any larvae which eat the leaves of untreated plants.
Only two groups of proteins have been proposed as commercially feasible products to control insects, namely the protein of this disclosure, dimercash, and three or four proteins isolated from cultures of the bacterium Bacillus thuringiensis. The physical and biological properties of these two classes of proteins are different as are their physiological effects on insects.
The principal protein from B. thuringiensis has a molecular weight of about 27,000 Daltons, occurs in the spore as a lipopolysaccharide protein and acts as a cytolytic endotoxin when the protein enters the gut of certain insects. Thus, this protein is a stomach poison and has no contact insecticidal actions in its present form. Crude preparations containing this protein have been used as commercial insecticides by dusting dried cultures of the organism onto the leaves of plants.
By contrast the protein, dimercash described in this disclosure, is a phosphorus containing protein; it is not a lipopolysaccharide protein. It has an amino acid composition which is similar to that of kappa casein. It has a dimer molecular weight of about 38,000 Daltons, is synthesized in mammary glands, and contains two phosphorus atoms.
The pure protein presents no suggestion of contact insecticidal activity when it is used alone; such action only appears when the protein is combined with a membrane transfer agent to form a synergistic mixture. The individual components by themselves show no action against insects at concentrations which are highly toxic when they occur together.
Thus, based on the molecular weights, the amino acid compositions, the sites of formation in cells and their actions on insects, B. thuringiensis proteins and the casein derived protein described in this disclosure are unrelated proteins having different physiological and toxicological actions. Their sole points of similarity are a) they both are relatively small proteins, b) they both are lipophilic and c) they both kill insects albeit by entirely different biochemical mechanisms.
The membrane transfer agents include detergents and solvents. Many of these if used in high concentrations may be toxic to insects. However, the proportions employed with dimercash do not visibly affect the insects. Moreover, the membrane transfer agents kill insects by an entirely different biochemical mechanism from that of the insecticidal compositions of this invention.
Insects killed by high concentrations of detergents, for example, die slowly, sluggishly and gently. Some researchers believe that detergents kill insects by blocking the respiratory pores, while others believe that the detergents affect the integrity of cell membranes. By contrast, insects killed by the synergistic mixture described herein, die in a frenzy of uncontrolled and uncoordinated activity. This type of death is typical of classic nerve poisons.
Some examples of commercial nerve toxins, which are toxic to insects, but relatively non-toxic to man, are malathion and parathion. These nerve poisons act by inhibiting the enzyme acetal choline esterase. This enzyme hydrolyzes the natural nerve stimulant, acetal choline, into inactive choline and acetic acid. If the enzyme is blocked by nerve poisons, then acetyl choline accumulates in the tissues and causes uncontrolled stimulation of muscles. This leads to frenzy of uncoordinated activity before death.
Whereas the classical phosphorous based nerve poisons block choline esterase by chemically reacting with serine amino acid at the active site on the choline esterase enzyme, the casein extract of this invention acts as a nerve toxin, when transferred across membranes, by an entirely different mode of action. It appears to affect either directly or indirectly the interaction of the acetyl choline with the receptor protein.